Promotion and inhibition of vesicle fusion by polylysine

Polylysine induced rapid aggregation of large unilamellar vesicles composed of phosphatidylcholine-cardiolipin (1:1 molar ratio) but not their fusion. Application of the terbium-dipicolinic acid fusion assay showed that addition of polylysine at nanomolar concentrations enabled a significant lowering of the Ca2+ threshold concentration for vesicle fusion from 9 to 1 mM. Analysis of the kinetics of fusion with a mass-action kinetic model showed that polylysine enhanced significantly the rate of aggregation but affected only slightly the rate of fusion per se. Maximal enhancement of overall fusion rates occurred at a charge ratio (polylysine/cardiolipin) of about 0.5. At larger polylysine concentrations, e.g., at charge ratios greater than 3, polylysine inhibited vesicle fusion.     

Network-Free Inference of Knockout Effects in Yeast

Perturbation experiments, in which a certain gene is knocked out and the expression levels of other genes are observed, constitute a fundamental step in uncovering the intricate wiring diagrams in the living cell and elucidating the causal roles of genes in signaling and regulation. Here we present a novel framework for analyzing large cohorts of gene knockout experiments and their genome-wide effects on expression levels. We devise clustering-like algorithms that identify groups of genes that behave similarly with respect to the knockout data, and utilize them to predict knockout effects and to annotate physical interactions between proteins as inhibiting or activating. Differing from previous approaches, our prediction approach does not depend on physical network information; the latter is used only for the annotation task. Consequently, it is both more efficient and of wider applicability than previous methods. We evaluate our approach using a large scale collection of gene knockout experiments in yeast, comparing it to the state-of-the-art SPINE algorithm. In cross validation tests, our algorithm exhibits superior prediction accuracy, while at the same time increasing the coverage by over 25-fold. Significant coverage gains are obtained also in the annotation of the physical network.     

Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse

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Highlights

• •Sufficient tumor tissues are often unavailable large HLA peptidome discovery.
• •Using patient derived xenograft (PDX) tumors can overcome this limitation.
• •The large PDX HLA peptidomes expand significantly those of the original biopsies.
• •The HLA peptidomes of the PDX tumors included many tumor antigens.

     

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16

Summary A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16q11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.     

Human acetylcholinesterase and butyrylcholinesterase are encoded by two distinct genes

1. Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities. 2. Homologous cosmid recombination with a 190-base pair 5' fragment from BuChEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3' end of BuChEcDNA from nervous system tumors and which has been mapped by in situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns. 3. The chromosomal origin of BuChE-encoding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BuChEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BuChEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringent in situ hybridization protocol, including washes at high temperature and low salt. 4. These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BuChEcDNA-hybridizing sequences, and present an example for a family highly similar proteins encoded by distinct, nonhomologous genes.     

Circadian variations in melatonin-binding sites in discrete areas of the male rat brain

The binding of 125I-melatonin to synaptosomes prepared from whole brains of male rats of the CD strain and from the brain, hypothalamus and striatum of male rats of the Sabra-Wistar strain was assessed throughout a 24 h period. The animals were maintained under a daily schedule of 14 h light (05:00-19:00 h) and 10 h darkness. In whole brain preparations the density of binding sites at 18:00 h was higher by about 70% than at 02:00 h with no variations in apparent affinity of the binding sites throughout the daily period. Specific binding of 125I-melatonin was found in both hypothalamus and striatum of the male rat with a distinct diurnal variation in binding site density in the hypothalamus only. The density of 125I-melatonin-binding sites in the hypothalamus was maximal between 10:00 and 18:00 h and dropped sharply after the lights went off. The apparent 125I-melatonin-binding affinities in these regions were constant and very similar to those in whole brain preparations. The daily variations in densities of 125I-melatonin-binding sites in discrete brain areas may represent a diurnal rhythmicity in the responsiveness of the neuroendocrine axis to melatonin.     

Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry

Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantitated from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners.     

Precipitation of calcium palmitate from bile salt-containing dispersions

Addition of calcium chloride to mixed micellar systems composed of sodium salts of palmitic acid and high concentrations of different bile acids results in precipitation of Ca(palmitate)2 only when the palmitate concentration exceeds a critical value, which is dependent on the concentrations of Ca2+, Na+ and bile salt, and on the type of bile salt used. All these dependencies, as well as the complex and interrelated effects of the various parameters on the kinetics of Ca(palmitate)2 precipitation are consistent with the following mechanism: (i) calcium binds to palmitate-bile salt mixed micelles and promotes their aggregation, at a rate governed by the concentration ratio between bound calcium and micelles (here denoted "binding ratio"). (ii) Ca(palmitate)2 precipitation occurs within the aggregate of micelles only if those micelles include sufficient amounts of Ca2+ and palmitate to allow for the formation of large enough crystal units of Ca(palmitate)2 which can serve as nucleation "seeds". Both the concentrations of micelles and Na+ have dual effects on the rate of precipitation. Increasing micelle concentration, by itself, accelerates aggregation but at the same time leads to a decrease of the binding ratio, thus reducing the rate of precipitation. Na+ which reduces the binding ratio through competitive binding also reduces the surface charge, thus assisting micelle aggregation. Our model also explains the facilitation of precipitation observed when phosphatidylcholine is contained in the palmitate-bile salt mixed micelles and the inhibitory effect of the water soluble bovine serum albumin.     

Carbon dioxide-independent and -dependent components of light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in oat leaves

Light inhibits while carbon dioxide enhances the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene in oat ( Avena sativa L. cv. Victory) leaf segments. The possibility that the light inhibition is mediated through changes of carbon dioxide has been investigated. The level of CO₂ increases or decreases in the sealed incubation vial in darkness or in light, respectively, which can apparently account for the differences in ACC-dependent ethylene production between the dark and light treatments. However, although the evolution of ethylene from ACC in the dark is reduced upon depletion of CO₂, the difference between light and dark is still very noticeable. Moreover, the production of the ethylene in CO₂-free air in the dark was still higher than in the light, where the concentration of CO₂ was 0.01%. It is proposed that the light effect on the conversion of ACC to ethylene is composed of two distinguishable components: one CO₂-mediated and the other CO₂-independent.